Protein disulfide isomerase (PDI) catalyzes the reversible formation and isomerization of disulfide bonds in proteins. This proposal focuses on two members of the PDI family-the traditional PDI, and a more recently discovered member of the PDI family, ERp57. We found that PDI mediates platelet aggregation, and intravascular PDI has been shown to be required for thrombus formation. We recently showed that ERp57 mediates platelet aggregation, hemostasis and thrombosis. ERp57 and PDI are involved in conversion of ?II3 to its high affinity state; however, the mechanisms by which they regulate ?II3 and platelet aggregation are unknown. Furthermore, there are now up to 20 different members of the PDI family, and a number of these are found in platelets. How these enzymes function together remains a mystery. Previous approaches have generally used non-specific inhibitors of PDI to document a role for PDI in platelet function and thrombosis. Newer approaches are therefore required to define the molecular roles of each enzyme, as well as the intravascular sources of these enzymes. We have generated targeted knockout mice with platelets specific deficiencies in ERp57 and in PDI, and transgenic mice with a mutant PDI. We have also generated an antibody to ERp57 that despite the high homology between ERp57 and PDI does not inhibit PDI. Our current goal is to characterize the role of intravascular and platelet-derived ERp57 in platelet function and thrombus formation. We will also characterize the role of platelet-derived PDI in platelet function and thrombosis. We hypothesize that platelets provide an essential source of these enzymes for hemostasis and thrombosis. The specific aims are to: 1. Characterize the role of intravascular and platelet-derived ERp57 in platelet accumulation and fibrin generation, and the role of platelet-derived ERp57 in platelet function; 2. Characterize the role of platelet-derived PDI in platelet function, thrombosis, platelt accumulation, and fibrin generation; and 3. Characterize the mechanism of activation of ?II3 by ERp57 and PDI. A principal technique used will be the laser-induced injury model of thrombosis. To determine the mechanisms by which ERp57 and PDI work, we will employ a thiol labeling strategy with mass spectrometry identification of the labeled thiols. We will determine the role of platelet-derived ERp57 and PDI in platelet function and thrombosis, and begin to unravel the mechanisms by which these enzymes work. Determining the extracellular redox mechanisms required for the final steps in the activation of ?II3 is a highly significant aspect of platelet function and thrombus formation that could lead to novel types of inhibitors or ways to regulate platelet aggregation.